Effects of Non-thermal Ultrasound on a Fibroblast Monolayer Culture: Influence of Pulse Number and Pulse Repetition Frequency.

Despite the use of therapeutic ultrasound in the treatment of soft tissue pathologies, there remains some controversy regarding its efficacy. In order to develop new treatment protocols, it is a common practice to carry out in vitro studies in cell cultures before conducting animal tests. The lack of reproducibility of the experimental results observed in the literature concerning in vitro experiments motivated us to establish a methodology for characterizing the acoustic field in culture plate wells. In this work, such acoustic fields are fully characterized in a real experimental configuration, with the transducer being placed in contact with the surface of a standard 12-well culture plate. To study the non-thermal effects of ultrasound on fibroblasts, two different treatment protocols are proposed: long pulse (200 cycles) signals, which give rise to a standing wave in the well with the presence of cavitation (ISPTP max = 19.25 W/cm2), and a short pulse (five cycles) of high acoustic pressure, which produces a number of echoes in the cavity (ISPTP = 33.1 W/cm2, with Pmax = 1.01 MPa). The influence of the acoustic intensity, the number of pulses, and the pulse repetition frequency was studied. We further analyzed the correlation of these acoustic parameters with cell viability, population, occupied surface, and cell morphology. Lytic effects when cavitation was present, as well as mechanotransduction reactions, were observed.

The Effect of PEGDE Concentration and Temperature on Physicochemical and Biological Properties of Chitosan.

Chitosan (CHT) is a polysaccharide with multiple claimed properties and outstanding biocompatibility, generally attributed to the presence of protonable amino groups rendering a cationic natural polymer. However, the effect of changes in CHT structure due to hydration is not considered in its performance. This study compares the effects on biocompatibility after drying at 25 °C and 150 °C scaffolds of chitosan, polyethylene glycol diglycidyl ether (PEGDE) crosslinked CHT (low, medium and high concentration) and glutaraldehyde (GA) crosslinked CHT. PEGDE crosslinked CHT showed a reduction in free amino groups and the amide I/II ratio, which exhaustive drying reduced further. In X-ray diffraction (DRX) analysis, PEGDE crosslinked CHT showed multiple peaks, whereas the crystallinity percentage was reduced with an increase in PEGDE concentration and thermal treatments at 150 °C. In a direct contact cell assay, high osteoblast viability was achieved at low and medium PEDGE concentrations, which was improved when the crosslinked scaffolds were thermally treated at 150 °C. This was attributed to its partial hydrophilicity, low crystallinity and low surface roughness; this in spite of the small reduction in the amount of free amino groups on the surface induced during drying at 150 °C. Furthermore, PEGDE crosslinked CHT scaffolds showed strong vinculin and integrin 1ß expression, which render them suitable for bone contact applications.

Production and characterization of supercritical CO2 dried chitosan nanoparticles as novel carrier device.

The materials produced by the supercritical CO2 drying have outstanding properties that allow the incorporation of molecules in their porous structure. In this context, dried chitosan nanoparticles including ß-lactoglobulin were obtained. First, the nanoparticles in water suspension were produced by ionotropic gelation incorporating the protein with high loading efficiency. Later, solvent exchange and CO2 supercritical drying procedures were performed. The physicochemical characteristics and structural properties were determined, demonstrating a stable porous structure in the dried materials and corroborating the presence of the protein after the drying. The CO2 supercritical dried chitosan nanoparticles can be effectively resuspended in acidic aqueous medium remaining in the nanoscale with minimum effect on the loading parameters. The release of the ß-lactoglobulin was highly influenced by the pH, reaching around 40% under acidic conditions in ten hours. The obtained results demonstrate the possibility to apply these chitosan materials as a controlled release material.